This invention relates to a method for the simultaneous and quantitative analysis of damaged white blood cells (WBC), nucleated red blood cells (NRBC) and white blood cell sub-populations (WBC/Diff). More particularly this invention relates to differentiating WBC, NRBC, damaged WBC a WBC subclass differential (WBC/Diff) in a whole blood sample by the use of multi-dimensional light scatter and fluorescence analysis and a lysing reagent capable of lysing red blood cells (RBC) without damaging WBC cellular membranes.
NRBC counts are conventionally determined by means of blood smear morphology. A stained blood smear is examined under the microscope and the NRBC are manually counted. In general, an NRBC concentration is reported as number of NRBC per 100 white blood cells ("WBC"). Normally, 200 WBC and the number of NRBC present in the same region on a patient blood smear are counted and the numbers are divided by 2 to express the NRBC concentration as the number of NRBC/100 WBC. The major drawback to this type of manual microscopic method is that it is very labor intensive, time-consuming, subjective and inaccurate due to poor statistics. Therefore, an accurate automated NRBC method has long been sought after by pathologists and laboratory technicians.
A major problem in automating a NRBC method for use on a clinical flow cytometer has been that since NRBC are rare events and RBC populations are so numerous, NRBC populations are not easily detected among the red blood cell ("RBC") population by either the differences in the cell's electrical resistivity (impedance measurements) or its light scattering characteristics (optical measurements). Although many attempts have been made to count NRBC among WBC populations, instead of among RBC population, these efforts have not generally been successful.
NRBC populations are not easily distinguished from WBC populations since NRBC do not form a well defined cluster among the WBC in the usual two dimensional space differentiation methods utilized on flow cytometers. One is usually not able to separate NRBC populations from the lymphocyte populations when the detected signals are viewed on the generally accepted, two-dimensional light scatter (forward vs. side) or light scatter vs. absorption, dot plots. The signals from the majority of the NRBC population is usually mixed in with the signals for RBC stroma and platelets ("PLT"), and the upper-end of NRBC cluster most often will extend into the space occupied by the lymphocyte population.
Automated clinical hematology instruments, such as the Technicon H*1.RTM., Coulter STK.RTM. S and Abbott Cell-Dyn.RTM. 3000 and 3500 instruments only "flag" samples for the possible presence of NRBC if the sample dot plot shows increased noise signals below the lymphocyte cluster. This type of flagging very often produces false positive results since the elevated noise level could be due to PLT clumps, giant PLT or incompletely lysed RBC. In addition, it is extremely difficult to obtain an accurate Total WBC count and WBC Differential ("WBC/Diff") on samples containing NRBC because of the interference. Additionally, blood smears of the flagged samples must be examined and counted under the microscope by a skilled technician to obtain accurate WBC differential and NRBC counts. This is a very labor-intensive and subjective process.
Notwithstanding these difficulties, the identification and quantitation of damaged cells among intact cells in a sample may be of importance for accurate characterization of cell populations exhibiting spontaneous cell death or cells affected by cytotoxic agents or cancer drugs. Nonviable cells may bind antibodies or other cellular markers non-specifically, and therefore should be identified and quantified in immuno-phenotyping as well as from hematology analysis.
In vivo, there are two different forms of cell death: apoptosis and necrosis. Apoptosis, the term introduced by Kerr et al., is a genetically programmed cell death which takes place during metamorphosis, embryogenesis, and morphogenesis. neutrophils undergo apoptosis during the inflammatory reaction, lymphocytes in the regulation of the immune system. Cell injury due to a variety of agents including chemotherapeutic cytotoxic insults may also lead to apoptosis. Apoptosis has also been demonstrated in premalignant and malignant tissues. During the process of apoptosis, the cell membrane remains intact and the cell breaks into apoptotic bodies which are then phagocytosed. Necrosis, or accidental cell death, on the other hand, occurs in response to harmful insults such as physical damage, hypoxia, hyperthermia, starvation, complement attack and chemical injury. These cells lose ability to selectively permeate extracellular materials and leak, finally losing their plasma membrane. Cells that have lost plasma membrane integrity become permeable to external compounds such dyes that normally will not penetrate the intact cell membrane are considered to be "nonviable" or damaged. Damaged cells which have lost their plasma membrane integrity also do not function metabolically.
In vitro, a similar phenomenon, cell death, occurs as a blood sample ages or during cell preparation procedures that damage the cell prior to flow cytometric analysis. Current cell preparation procedures subject the cells to a long process including labelling cells with monoclonal antibodies (Mab), lysing of red blood cells (RBC) and fixing of the white blood cells (WBC) to prevent further destruction.
The combination of 90.degree. and forward light scatter techniques have been used in the art to discriminate damaged cells from intact cells. It has been found however, that light scatter alone is not sensitive enough to clearly separate and quantitate the damaged or nonviable cells from the viable or intact cells. This is particularly true if a sample contains heterogenous cell populations such as a blood sample. Use of a fluorescent nucleic acid stain in addition to multi-angle light scatter dramatically increases the sensitivity of the detection for dead cells.
Currently a variety of techniques exist utilizing light scatter and fluorescence techniques for determining whether a cell in a sample is intact or damaged. According to the art viable, intact cells can be distinguished from dead cells by using either fluorescein diacetate (FDA) or propidium iodide (PI). In these methods, the sample is treated with either FDA or PI. The cells which stain with FDA are considered viable and the cells which stain with PI are considered "dead". However, these methods are limited in that the cells cannot be fixed since fixed cells generate autofluorescence. In addition, fluorescein, which is a product of FDA post hydrolysis by intracellular esterase, is so bright that it overwhelms the immunofluorescence signals from other stains such as FITC or phycoerythrin (PE).
U.S. Pat. Nos. 4,661,913 and 4,284,412 describe methods of differentiating WBC subpopulations by light scatter analysis on a flow cytometer. U.S. Pat. No. 4,520,110 describes a method of differentiating heterogenous leukocyte populations by immuno-phenotyping using a combination of light scatter and fluorescence. Each of the above described methods require manual sample preparation and incubation time much too long to be incorporated on a rapid multi-parameter hematology analyzer of today. Additionally, these references do not appear to teach how to discriminate damaged cells from intact cells.
U.S. Pat. No. 4,751,188, to Valet, describes a method which is based on the principle that cellular components can be stained by dyes and measured simultaneously with cell volume, for example in a flow cytometer. According to the patent a complete blood count can be produced within a few minutes. In this method, a blood sample is manually prepared by a procedure that comprises the steps of: making a 1:250 dilution of the sample with buffered isotonic saline; adding 5 microliters of a predetermined concentration of a stock solution which contains a fluorescent RNA/DNA stain, a fluorescent membrane-potential-sensitive stain, fluorescing monodisperse calibration particles and an organic solvent in which the dyes dissolve; and incubating the mixture at room temperature for 3 to 5 minutes. The dyes are stored in an organic solvent such as DMSO or DMF as a stock solution and only a very small amount of these stock dye solutions are added directly to the cell suspension to stain the cells. The prepared cell suspension is then aspirated through flow cytometer flow cell for measurement. In this method, a DNA/RNA stain is used. This DNA/RNA stain is either acridine orange (AO), quinacrine (QA), or pyonine Y (PY) and the membrane-sensitive stain is 3,3-dehexyl-oxacarbocyanine (DiOC6). Additionally, at least one additional stain is used, being selected from the group of: cell protein stains; lipid stains; enzyme stains; intracellular pH stains; and SH group stains. The methodology of Valet, as described in this patent: 1) is not fully automatable because of the step required for manual addition of a very small volume of dye dissolved in organic solvents; 2) is relatively long, as the amount of time necessary to complete the blood cell counts; 3) requires at least two dyes to characterize the blood cell this may produce a problem of quenching; 4) the requirement of making a 250 fold dilution of a blood sample does not provide enough WBC's to produce statistically satisfactory results unless the counting time is much prolonged; 5) does not demonstrate that it is possible to separate monocytes, eosinophils and basophils, suggesting that the teachings of Valet can only produce an incomplete WBC differential results (only two WBC subpopulations, granulocytes and lymphocytes, are shown in the figures and examples).
U.S. Pat. No. 5,057,413, to Terstappen, discloses a flow cytometric method for discriminating between intact and damaged cells. In this method both intact and damaged cells in a sample are stained. The teachings of Terstappen are based upon the principle that there is a sufficient difference in the fluorescent intensity of the stained intact cells and that of damaged cells. A further stated objective of the disclosure is to use the differentiation methods of the art in conjunction with monoclonal antibodies (Mabs) fluorescently labelled with FITC or PE to simultaneously identify cellular antigens, intact cell and damaged cells, wherein the peak emission spectra of each fluorescent label must be distinguishable from each other and from a nucleic acid dye. In this method, RBC's are lysed with ammonium chloride for 3 to 5 minutes, and centrifuged at 200 g for 5 minutes. The pellet was washed twice with RPMI 1640 culture medium, each time centrifuging at 200 g for 5 minutes. And then the cells were resuspended in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA). When Mabs are added to the sample, incubated 20 minutes on ice, the cells were washed twice with the PBS solution and the cells were resuspended in 1 ml of 1% paraformaldehyde in PBS. A stock solution of LDS-751 was made in methanol and the working solution was prepared by diluting the stock solution in PBS. Ten microliters of this working solution is added to the prepared cell suspension. In another experiment, unfixed WBC's, post ammonium chloride lyse of RBC's, were resuspended and kept in RPMI solution for 1 hour before analysis in order to obtain optimal light scattering properties of the cells.
The Terstappen method has several problems in that may variable in the fixation process, such as temperature, concentration of the fixative(s) and the duration of the fixation, can change the permeability of the cell membrane and thus the intensity of the staining. The hard fixed, originally intact cells may have the same staining intensity as that of the hard fixed, damaged cells since the DNA content of all cells of the same individual is the same (proliferating hyperploidy tumor or leukemic cells are exceptions). In addition, if any DNA fragmentation occurs at the late stage of cell death, then the damaged cells will contain less DNA and the staining intensity will decrease. The Terstappen method is also long and cumbersome making it an difficult, if not impossible to incorporate onto a fully automated hematology instrument of today. The results reported in the patent also indicate that a large portion of the damaged cells may have occurred during the required long and torturous sample preparation procedure.
Recently, U.S. Pat. No. 5,298,426, issued on Mar. 29, 1994, to Inami et al. for the detection of NRBC. This patent teaches a two-step method comprising the staining of WBC and NRBC by specific nuclear stains. In this patented method, a blood sample is first mixed with an acid hypotonic solution containing a fluorescent nuclear dye. Then, a solution comprising an alkaline salt buffer, to adjust pH and Osmolarity, is mixed with the sample/first reagent solution. This final solution is then loaded into a flow cytometer to detect and count NRBC along with other nucleated cells.
There are several reasons why the Inami et al. approach is not acceptable, especially for an automatable method. First, an acidic-hypotonic solution damages all cell membranes making the WBC leaky and therefore selective staining of NRBC nuclei by a nuclear stain is not possible. There are no known dyes which stain only NRBC nuclei and not WBC nuclei since the nuclear material (DNA) is the same. The nuclear stain claimed by Inami et al., is Propidium Iodide, a commonly used nucleic acid stain.
Additionally, the Inami et al. method does not separate or distinguish the fluorescent signals of the NRBC nuclei from that of other nuclear remnants such as Howell-Jolly Bodies, Basophilic Stippling, RNA from lysed reticulocytes and reticulated platelets, and DNA from WBC and Megakaryocytic fragments. Third, the Inami et al. method requires that the sample be pretreated, off-line, using several reagents to "prep" the sample before the prepped sample/reagent solution can be loaded into the instrument.
The problems in the existing art described above have been resolved in the present invention.
Accordingly, an object of the present invention is to provide an accurate method of distinguishing damaged WBC's from intact WBC's, and for rapidly quantitating the WBC differential (WBC/Diff), NRBC's, and damaged WBC's in a whole blood sample.
Another objective of the present invention is to provide a fully automatable method for distinguishing damaged WBC's from intact WBC's, and for rapidly quantitating the WBC/Diff, NRBC's, and damaged WBC's in a whole blood sample.
Yet another object of the present invention is to provide an automated method for distinguishing damaged WBC's from intact WBC's, and for rapidly quantitating the WBC/Diff, NRBC's, and damaged WBC's in a whole blood sample and for immuno-phenotyping.